One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as protein phosphatases. It is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
Nearly all cell surface receptors use one or more of the mitogen-activated protein kinase (MAP kinase) cascades during signal transduction. Three distinct subgroups of the MAP kinases have been identified and each of these consists of a specific module of downstream kinases. One subgroup of the MAP kinases is the Jun N-terminal kinase/Stress activated protein kinase (JNK/SAPK) cascade. This pathway was originally identified as an oncogene- and ultraviolet light-stimulated kinase pathway but is now known to be activated by growth factors, cytokines and T-cell costimulation (Widmann et al., Physiol. Rev., 1999, 79, 143-180).
MEKK2 (also known as mitogen-activated protein kinase kinase kinase 2, MEK kinase 2 and MAP/ERK kinase kinase 2) is a dual specific serine/threonine kinase that functions to mediate cellular responses to mitogenic stimuli. The MEKK2 protein has been shown to regulate signaling events associated with two of the three branches of MAP kinase pathways.
MEKK2 was originally isolated and cloned from mouse NIH3T3 cells and only recently has the human sequence been identified. Therefore a majority of studies on MEKK2 have been performed using the mouse MEKK2 sequence transfected into human cell lines. Disclosed in U.S. Pat. No. 5,854,043 are the nucleic acid and protein sequence of murine MEKK2 as well as regulatory and catalytic domains of the protein (Johnson, 1998). Expression of murine MEKK2 transfected into HEK293 cells, a human embryonic kidney cell, was shown to activate the JNK/SAPK and p42/44 MAPK pathways (Blank et al., J. Biol. Chem., 1996, 271, 5361-5368). Within these pathways, MEKK2 was shown to phosphorylate JNK kinase and MEK1, each a downstream kinase of the JNK/SAPK and p42/44 MAPK pathways, respectively (Blank et al., J. Biol. Chem., 1996, 271, 5361-5368; Deacon and Blank, J. Biol. Chem., 1997, 272, 14489-14496).
MEKK2 has been shown to interact with 14-3-3 proteins via the kinase domain in the absence of any phosphorylation events. 14-3-3 proteins are a group of acidic proteins thought to play a role in controlling mitogenic signal transduction pathways. In these studies it was demonstrated that MEKK2 interacts with 14-3-3 proteins, and this protein--protein interaction may represent a non-enzymatic mechanism for controlling the effects of MEKK2 in the MAP kinase pathway (Fanger et al., J. Biol. Chem., 1998, 273, 3476-3483).
In transfected COS cells, MEKK2 protein was localized primarily to Golgi-associated structures, with a component of MEKK2 being found in the cytoplasm. These same studies also found that MEKK2 is activated by epidermal growth factor (EGF)(Fanger et al., Embo J., 1997, 16, 4961-4972).
Recently MEKK2 has been implicated in inflammatory responses. Zhao et al. have shown that MEKK2 can activate the NF-kappa-B pathway in HeLa cells. NF-kappa-B is a transcription factor that translocates to the nucleus affecting the transcription of several genes upon cellular induction by proinflammatory agents. MEKK2 was shown to induce NF-kappa-B activity by phosphorylating an inhibitor molecule, IkB, that sequesters NF-kappa-B in the cytoplasm. This phosphorylation releases NF-kappa-B for translocation into the nucleus (Zhao and Lee, J. Biol. Chem., 1999, 274, 8355-8358).
The pharmacological modulation of MEKK2 activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of MEKK2 and to date, investigative strategies aimed at modulating MEKK2 function have involved the use of antibodies and molecules that block upstream entities or kinase pathways in general. Disclosed in the PCT application WO 98/54203 are inhibitors of the SAPK pathway comprising antisense oligonucleotides to an upstream kinase, MEKK1, as well as antisense oligonucleotides targeting the kinase isoforms that lie downstream of JNKK1, SAPK1-3. Also disclosed are methods to inhibit the SAPK pathway, of which MEKK2 is a member, using ribozymes, small molecule inhibitors and dominant-negative mutants (Mercola, 1998). Methods of treating allergic inflammation in humans by defining molecules that interfere with the MEKK/JNKK-contingent signal transduction pathway are disclosed in U.S. Pat. No. 5,910,417. Further disclosed are methods of using hematopoietic cells to screen for said molecules (Gelfand and Johnson, 1999).
However, these strategies are untested as therapeutic protocols as well as being non-specific to MEKK2. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting MEKK2 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of MEKK2 expression.
The present invention provides compositions and methods for modulating MEKK2 expression.